Notably, upon exposure to tumor necrosis factor- and -interferon, PECAM-1 leaves endothelial cell junctions and redistributes diffusely onto the cell surface (Romer et al. resulted in a marked increase in both adherence and spreading of PECAM/FKBP-2Ctransfected cells on immobilized fibronectin, a reaction that was mediated by the integrin 51. These data demonstrate that signals MS-444 required for integrin activation can be elicited by clustering of MS-444 PECAM-1 from inside the cell, and suggest that a dynamic equilibrium between PECAM-1 monomers, dimers, and oligomers may control cellular activation signals that influence the MS-444 adhesive properties of vascular cells that express this novel member of the immunoreceptor tyrosineCbased inhibitory motif family of regulatory receptors. Triton-soluble fraction was analyzed under both reducing and nonreducing conditions on a 7% SDSCpolyacrylamide gel. After transfer to an Immobilion-P membrane (Millipore), proteins were visualized using antibodies to PECAM-1 (PECAM-1.3) or FKBP-12 in conjunction with enhanced chemiluminescence immunodetection (Amersham Pharmacia Biotech) MS-444 using peroxidase-conjugated goat antiCmouse IgG (Jackson ImmunoResearch Laboratories). For immunoprecipitation analysis, detergent cell lysates were prepared as above, and the Triton-soluble fractions were incubated with 10 g/ml PECAM-1.3 IgG overnight at 4C. Immune complexes were captured with 50 l of 50% slurry of protein GCSepharose for 1 h at 4C, and then washed five times with immunoprecipitation wash buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, and 2% Triton X-100). Bound proteins were eluted from the beads by boiling for 10 min in 30 l of SDS-reducing buffer and analyzed by SDS-PAGE, as described above. Flow Cytometry 4 g of PECAM-1.3 or PECAM-1/IgG in 100 l of Rabbit Polyclonal to GRIN2B HPBS was added to 5 105 wild-type or transfected HEK-293 cells in 100 l of HPBS and allowed to incubate for 60 min at room temperature in the presence or absence of various concentrations of AP1510. The cells were then washed by centrifugation with 1 ml of HPBS and then resuspended in 100 l of HPBS containing 1 g of FITC-labeled goat antiChuman or goat antiCmouse IgG chain (Jackson ImmunoResearch Laboratories). After a 30 min incubation at 4C, the cells were washed once more in HPBS, and then subjected to flow cytometric analysis. Fluorescence Microscopy Wild-type or stably transfected 293 cells were incubated in the presence or absence of 1 M AP1510 for 30 min at room temperature, and then by 5 g/ml Alexa 488Cconjugated PECAM-1.3 for 30 min at 4C. The cells were then washed, fixed with 1% formaldehyde, and mounted onto glass slides using a cytospin preparation. The cell surface distribution of PECAM-1 was analyzed using a Zeiss fluorescent microscope (ZEISS). Quantitative Evaluation of the Binding of 293 Cells To Immobilized Fibronectin Adherent 293 cells were lifted using 0.01% trypsin and 10 mM EDTA, washed in sterile HPBS, and resuspended at 106 cells/ml in prewarmed (37C) serum-free medium. After incubation with 2 g/ml calcein AM (Molecular Probes) for 30 min at 37C, cells were washed and resuspended at 106 cells/ml in prewarmed serum-free medium, and then incubated again, this time with varying concentrations of AP1510 in the presence of selected antibodies for 30 min at 22C. 200-l cell aliquots were then added to wells of 96-well microtiter tissue culture plates (Dynatech Laboratories) that had been coated with either 10 g/ml fibronectin (Sigma-Aldrich) or 10 mg/ml BSA (ICN Biomedicals). Antibody competition experiments were performed by preincubating cells with varying concentrations of selected integrin-specific mAbs for 30 min at 22C, before addition of cells to the wells. The reaction was stopped by addition of 70 l of 4% formaldehyde for 15 min at 37C, and total well fluorescence was determined in a CytoFluor fluorescent plate reader (PerSeptive Biosystem). MS-444 Loosely adherent and unbound cells were then removed by washing the wells with prewarmed serum-free medium, and fluorescence was measured once more to determine percent cell adhesion. Results PECAM-1 Exists in an Equilibrium between Monomeric, Dimeric, and Oligomeric Forms Within the Plasma Membrane Previous studies have shown that antibody-mediated dimerization or oligomerization of PECAM-1 can lead to upregulation of integrin function in a variety of different cell types. Whether PECAM-1 actually forms lateral associations within the plane of the plasma membrane and whether these associations are able to influence the.